User:Behzad Damadzadeh/Notebook/PcTF Subcloning in E-coli/2013/06/06

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(Autocreate 2013/06/06 Entry for User:Behzad_Damadzadeh/Notebook/PcTF_Subcloning_in_E-coli)
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=Date=
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==06/06/2013==
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<!-- Precede each finished task with a checkmark &#x2713; -->
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* &#x2713; Minipreps: FlpE (4)
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'''List title'''
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----
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# List items
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'''Minipreps'''<br>
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> Check with E, P, and E/P digests
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{| {{table}} cellspacing="7"  width=700px
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|-valign="top"
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| <u>Reagent</u> || <u>1,4,7,10</u> || <u>2,5,8,11</u> || <u>3,6,9,12</u>
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| rowspan="7" | <u>Expected:</u><br>1. FlpE, E = 7160, 546<br>2. FlpE, P = 7706<br>3.FlpE, E/P=5649, 1511, 546
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| rowspan="7" | [[Image:Flp-E.jpg|300px|E/P digests 05/06/10]]<br>15 μL/lane; 1% agarose
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|-
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| DNA(plasmid) || 2.0 || 2.0 || 2.0
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|-
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| 10X buffer || 1.5 || 1.5 || 1.5
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|-
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| EcoRI || 1.0 || --- || 1.0
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|-
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| PstI || --- || 1.0 || 1.0
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|-
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| dH<sub>2</sub>O || 10.5 || 10.5 || 9.5
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|-
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| &nbsp;<br>&nbsp;<br>&nbsp;<br>&nbsp;||
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|}
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15 μL --> 37°C/ ~15 min.
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--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)<br>
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--> Streak clone (saved toothpick) onto 100 μg/mL Amp plate.<br>
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<!-- Precede each finished task with a checkmark &#x2713; -->
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* &#x2713; Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
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* &#x2713; Polycomb-ATF negative control lines: colony plates
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----
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'''1 day before transfection:'''<br>
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# Seed cultures in 6-well plates at ~2.5x10<suP>5</sup> so that cells will be ~5.0x10<sup>5</sup> at the time of transfection.
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'''Transfections'''<br>
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> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)<br>
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--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in<br>
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> Use Lipofectamine, 6-well format<br>
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> Plates:
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# BD002 (3:1) Flp-in T-REx
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#BD002 (4:1) Flp-in T-REx
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BD002 (3:1) = 339 ng/μl
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BD002 (4:1) = 317 ng/μl
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KAH187 = 441.5 ng/μl
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FlpE = 192 ng/μl
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{| {{table}} cellspacing="7"  width=700px
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|-
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| Plate-Well || Plasmid || DNA || Volume|| dH2O|| Lipo  || PLUS reagent || <u>Opti-MEM (total)</u>
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|-
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| 1-1            || BD002 (3:1) + FlpE  || 1.5 + 0.5 μg || 4.42 + 2.60 μL || 12.98μL|| 2.5μL|| 7.5 μL      || 570 μL
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|-
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| 1-2            || BD002 (4:1) + FlpE || "          || 4.73 + 2.60 μL  || 12.67μL|| "  ||    "      || "
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|-
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| 1-3 (-) Ctrl    || BD002 (3:1) + --- || 2 μg      || 4.42 + --- μL  || 15.58μL || "    ||  "    || "
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|-
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| 1-4 (-)Ctrl      ||  --- + --- || 0 μg        || --- + --- μL  || 20.0μL|| "    ||    "    || "
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|-
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|1-5            || KAH187    ||  20 μg        ||  4.53  || 15.46μL||    "  || "||"
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|}
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# Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
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## Label sterile microfuge (1.5 ml) tubes.
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## Add '''570 μL Opti-MEM''' to each 20 μL DNA sample.
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## Add '''2.5 μL PLUS reagent''' to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
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## Add '''7.5 μL Lipofectamine LTX''' to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
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# Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture '''drop-wise''' to each appropriate well of cells.
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# Incubate cells at 37°C in a CO<sub>2</sub> incubator
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# (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.
 +
 
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Transgene expression should be detectable after 18 hours.
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 +
 
 +
 
 +
----
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<!-- ##### DO NOT edit below this line unless you know what you are doing. ##### -->
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__NOTOC__

Current revision

PcTF Subcloning in E. coli Main project page
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06/06/2013

  • ✓ Minipreps: FlpE (4)

Minipreps
> Check with E, P, and E/P digests

Reagent 1,4,7,10 2,5,8,11 3,6,9,12 Expected:
1. FlpE, E = 7160, 546
2. FlpE, P = 7706
3.FlpE, E/P=5649, 1511, 546
E/P digests 05/06/10
15 μL/lane; 1% agarose
DNA(plasmid) 2.0 2.0 2.0
10X buffer 1.5 1.5 1.5
EcoRI 1.0 --- 1.0
PstI --- 1.0 1.0
dH2O 10.5 10.5 9.5
 
 
 
 

15 μL --> 37°C/ ~15 min.


--> Conclusion: Success! Combine 4 preps (300μL total) for DNA clean-up (tomorrow)
--> Streak clone (saved toothpick) onto 100 μg/mL Amp plate.


  • ✓ Transfections: H3K27me3 reporters into cell lines BD002 (KAH201+KAH182+V0200)
  • ✓ Polycomb-ATF negative control lines: colony plates



1 day before transfection:

  1. Seed cultures in 6-well plates at ~2.5x105 so that cells will be ~5.0x105 at the time of transfection.

Transfections

> U2OS Flp-in T-REx lines (puro/zeo/blast) + FRT-marked mammalian transfection vector (hygro)
--> Note: co-transfect with FlpE (1:3); hygro resistance replaces zeo resistance after "Flp" in
> Use Lipofectamine, 6-well format
> Plates:

  1. BD002 (3:1) Flp-in T-REx
  2. BD002 (4:1) Flp-in T-REx

BD002 (3:1) = 339 ng/μl BD002 (4:1) = 317 ng/μl KAH187 = 441.5 ng/μl FlpE = 192 ng/μl

Plate-Well Plasmid DNA Volume dH2O Lipo PLUS reagent Opti-MEM (total)
1-1 BD002 (3:1) + FlpE 1.5 + 0.5 μg 4.42 + 2.60 μL 12.98μL 2.5μL 7.5 μL 570 μL
1-2 BD002 (4:1) + FlpE " 4.73 + 2.60 μL 12.67μL " " "
1-3 (-) Ctrl BD002 (3:1) + --- 2 μg 4.42 + --- μL 15.58μL " " "
1-4 (-)Ctrl --- + --- 0 μg --- + --- μL 20.0μL " " "
1-5 KAH187 20 μg 4.53 15.46μL " ""
  1. Prepare DNA-Lipofectamine complexes (600 μl per sample) as follows:
    1. Label sterile microfuge (1.5 ml) tubes.
    2. Add 570 μL Opti-MEM to each 20 μL DNA sample.
    3. Add 2.5 μL PLUS reagent to each DNA+Opti-MEM sample. Incubate for 5 minutes at room temperature.
    4. Add 7.5 μL Lipofectamine LTX to each DNA+Opti-MEM+PLUS reagent sample. Incubate for 30 minutes at room temperature.
  2. Add the total 600 μL of DNA+Opti-MEM+PLUS reagent+Lipofectamine mixture drop-wise to each appropriate well of cells.
  3. Incubate cells at 37°C in a CO2 incubator
  4. (Optional) To reduce toxicity, after 5-6 hours wash cells once with warm 1xPBS to remove excess DNA-Lipo complexes. Add fresh antibiotic-free growth medium.

Transgene expression should be detectable after 18 hours.




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